Each beat of the heart begins as a spontaneous electrical depolarization of specialized pacemaker cells in the sinoatrial node. The sympathetic nervous system increases heart rate primarily by releasing norepinephrine from nerves that innervate the sinoatrial node. Within the sinoatrial myocytes, this aspect of the sympathetic fight-or-flight response requires communication between the 2 adrenergic receptors (2ARs) that respond to the norepinephrine and the ion channels that collectively control the timing and shape of action potentials. The long-term goal of this project is to understand the molecular machinery that produces and regulates pacemaker activity in sinoatrial myocytes. Experiments outlined in this proposal focus on some aspects of signaling between 2ARs and hyperpolarization-activated, cyclic nucleotide sensitive (HCN, or pacemaker) ion channels. HCN channels are activated by 2ARs and are thought to be critical both for setting the resting heart rate and for mediating the positive chronotropic effect of 2 agonists. However, the biophysical mechanisms for HCN channel involvement in pacemaking, and the functional and physical relationships between HCN channels and 2 adrenergic receptors are poorly understood. The working hypotheses to be tested in this project are that a leak current produced by HCN channels is critical for pacemaker activity in sinoatrial myocytes, and that sympathetic regulation of pacemaking requires a macromolecular signaling complex that contains 2ARs and HCN channels. These questions will be addressed using expressed HCN channels, acutely isolated murine sinoatrial myocytes and cultured sinoatrial myocytes. The principle techniques to be employed are patch clamp electrophysiology, confocal immunofluorescent microscopy, and immunoaffinity chromatography. There are three specific aims: (1) To understand the biophysical mechanisms for HCN channel activity during diastole, (2) To describe the functional relationships between 2ARs and HCN channels that control firing rate in sinoatrial myocytes, and (3) To determine the subcellular localization and physical interactions of proteins that participate in sympathetic control of pacemaking. [unreadable] [unreadable] [unreadable]